JPWO2010093014A1 - Plant extract containing ice crystallization inhibitor and method for producing the same - Google Patents
Plant extract containing ice crystallization inhibitor and method for producing the same Download PDFInfo
- Publication number
- JPWO2010093014A1 JPWO2010093014A1 JP2010550559A JP2010550559A JPWO2010093014A1 JP WO2010093014 A1 JPWO2010093014 A1 JP WO2010093014A1 JP 2010550559 A JP2010550559 A JP 2010550559A JP 2010550559 A JP2010550559 A JP 2010550559A JP WO2010093014 A1 JPWO2010093014 A1 JP WO2010093014A1
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- Prior art keywords
- plant
- extract
- ice crystallization
- ice
- crystallization inhibitor
- Prior art date
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Classifications
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- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- Life Sciences & Earth Sciences (AREA)
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- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Fruits And Vegetables (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Peptides Or Proteins (AREA)
- Compounds Of Unknown Constitution (AREA)
Abstract
本発明の解決課題は、実用にかなう優れた氷結晶化阻害活性を有する氷結晶化阻害物質を、食品製造に利用できる安全な工程で、簡単に、効率よく、安価に提供することである。本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第一方法は、氷結晶化阻害物質を含む植物を乾燥する工程;および、乾燥した植物から氷結晶化阻害物質を抽出する工程を含むことを特徴とする。また、本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第二方法は、植物から氷結晶化阻害物質を抽出する工程;および、抽出物を吸着体で処理する工程を含むことを特徴とする。An object of the present invention is to provide an ice crystallization inhibitor having an excellent ice crystallization inhibitory activity suitable for practical use in a simple, efficient and inexpensive manner in a safe process that can be used for food production. A first method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of drying a plant containing an ice crystallization inhibitor; and an ice crystallization inhibitor is extracted from the dried plant. Including the step of: The second method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of extracting an ice crystallization inhibitor from a plant; and a step of treating the extract with an adsorbent. It is characterized by including.
Description
本発明は、氷結晶化阻害物質を含む植物抽出物およびその製造方法に関するものである。 The present invention relates to a plant extract containing an ice crystallization inhibitor and a method for producing the same.
生体における低温時の防御物質として、植物、魚類、昆虫、菌類、細菌等が産生する氷結晶化阻害物質が発見されている。これらの氷結晶化阻害物質は、霜害防除剤、冷凍食品の品質向上、細胞や組織等の生体材料の保存、低温手術での利用等において有用であるとされている。 Ice crystallization inhibitors produced by plants, fish, insects, fungi, bacteria and the like have been discovered as defense substances at low temperatures in living bodies. These ice crystallization inhibitors are said to be useful in frost damage control agents, quality improvement of frozen foods, preservation of biological materials such as cells and tissues, and use in cryosurgery.
氷結晶化阻害物質を含む生物として、例えば、植物としてはイネ科の植物や(非特許文献1〜3)、ダイコン等のアブラナ科の植物(特許文献1〜3)が知られている。これらの植物由来の氷結晶化阻害物質は、その含量が少なく、実用化において製造方法に課題がある。また、魚類では、例えば、カジカ科等の魚類、昆虫類ではゴミムシダマシの幼虫に由来する氷結晶化阻害物質が知られている(非特許文献4〜6,特許文献4)。しかしながら、魚類又は昆虫類由来の氷結晶化阻害物質を工業的に生産することは困難である。それは、大量調製するために必要な多くの個体数を集めることが困難であり、また、硬い骨格等を持つため精製に高いコストを要することが大きな課題となっているからである。 Known organisms containing an ice crystallization inhibitor include, for example, grass plants (Non-patent Documents 1 to 3) and cruciferous plants such as Japanese radish (Patent Documents 1 to 3). These plant-derived ice crystallization inhibitors have a low content, and there is a problem in the production method in practical use. In fish, for example, ice crystallization inhibitory substances derived from larvae of Bacillus terrestris are known for fish such as scorpionidae and insects (Non-patent Documents 4 to 6, Patent Document 4). However, it is difficult to industrially produce ice crystallization inhibitors derived from fish or insects. This is because it is difficult to collect a large number of individuals necessary for large-scale preparation, and since it has a hard skeleton and the like, a high cost is required for purification.
このように、これまでに報告されている氷結晶化阻害物質を含む植物、魚類、昆虫、菌類、細菌等は、生体内に氷結晶化阻害物質を微量しか含まず、その抽出効率が非常に悪かったり、多量に含むものであっても捕獲や培養が難しいといった問題がある。よって、これまでこれら生物から氷結晶化阻害物質を工業的に生産し、食品用途として利用することはできなかった。魚類又は昆虫類の氷結晶化阻害物質については、遺伝子組み換え技術を用いて生産性を高めている報告もある(特許文献5,6)。しかしながら、そのような組換え技術を利用しなくとも、より簡便な方法で効率よく、安価に氷結晶化阻害物質を提供し得る方法が求められている。 As described above, plants, fish, insects, fungi, bacteria, and the like containing ice crystallization inhibitors reported so far contain only a very small amount of ice crystallization inhibitors in the living body, and their extraction efficiency is very high. Even if it is bad or contains a large amount, it is difficult to capture and culture. Therefore, until now, it has not been possible to industrially produce an ice crystallization inhibitor from these organisms and use it for food. There is also a report of improving the productivity of fish or insect ice crystallization inhibitors using genetic recombination techniques (Patent Documents 5 and 6). However, there is a need for a method that can provide an ice crystallization inhibitor substance efficiently and inexpensively by a simpler method without using such a recombinant technique.
氷結晶化阻害物質を含有する組成物から、不要物を除去して目的の氷結晶化物質を得る方法としては、例えば限外濾過による分画法(特許文献7,8)や、各種のクロマトグラフィーによる精製法(特許文献2,8)、有機溶媒を用いた分画法(特許文献2,9)についての報告がある。しかしながら、これらの工程には特別な設備や装置が必要であり高いコストを要することが課題であり、また、特に限外濾過やクロマトグラフィーを用いた分画は完了までに長時間を要するなどの問題がある。 Examples of a method for obtaining an intended ice crystallization substance by removing unnecessary substances from a composition containing an ice crystallization inhibitor include a fractionation method by ultrafiltration (Patent Documents 7 and 8), various chromatographies, and the like. There are reports on a purification method by graphy (Patent Documents 2 and 8) and a fractionation method using an organic solvent (Patent Documents 2 and 9). However, these processes require special equipment and equipment and require high costs. In particular, fractionation using ultrafiltration or chromatography takes a long time to complete. There's a problem.
本発明の解決課題は、実用にかなう優れた氷結晶化阻害活性を有する氷結晶化阻害物質を、食品製造に利用できる安全な工程で、簡単に、効率よく、安価に提供することである。 The problem to be solved by the present invention is to provide an ice crystallization inhibitor having an excellent ice crystallization inhibitory activity suitable for practical use, simply, efficiently and inexpensively in a safe process that can be used for food production.
本発明者らは、上記課題を解決するために鋭意検討を行なった。その結果、氷結晶化阻害物質を含む植物を乾燥処理することによりその植物が有する氷結晶化阻害活性を著しく向上させることができること、また、氷結晶化阻害物質を含んだ植物抽出液を吸着体で処理することにより抽出液中の不要分を選択的に除去し、氷結晶化阻害物質の精製度を著しく向上させることができることを見出し、本発明を完成させた。 The present inventors have intensively studied to solve the above problems. As a result, it is possible to remarkably improve the ice crystallization inhibitory activity of the plant by drying the plant containing the ice crystallization inhibitor, and to adsorb the plant extract containing the ice crystallization inhibitor. As a result, it was found that the unnecessary components in the extract can be selectively removed by the treatment with the above, and the purification degree of the ice crystallization inhibitor can be remarkably improved, and the present invention has been completed.
本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第一方法は、氷結晶化阻害物質を含む植物を乾燥する工程;および、乾燥した植物から氷結晶化阻害物質を抽出する工程を含むことを特徴とする。 A first method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of drying a plant containing the ice crystallization inhibitor; and an ice crystallization inhibitor is extracted from the dried plant. Including the step of:
本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第二方法は、植物から氷結晶化阻害物質を抽出する工程;および、抽出物を吸着体で処理する工程を含むことを特徴とする。 A second method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of extracting an ice crystallization inhibitor from a plant; and a step of treating the extract with an adsorbent. It is characterized by.
本発明に係る植物抽出物は、上記の本発明に係る第一方法または第二方法により得られる植物抽出物であって、氷結晶化阻害活性を有することを特徴とする。 The plant extract according to the present invention is a plant extract obtained by the first method or the second method according to the present invention, and has an ice crystallization inhibitory activity.
また、本発明に係る氷結晶化阻害物質含有組成物は、上記植物抽出物、または上記植物抽出物の乾燥物を含むことを特徴とする。 Moreover, the composition containing an ice crystallization inhibitor according to the present invention contains the above plant extract or a dried product of the plant extract.
本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第一方法は、氷結晶化阻害物質を含む植物を乾燥する工程;および、乾燥した植物から氷結晶化阻害物質を抽出する工程を含むことを特徴とする。以下、当該方法を工程ごとに説明する。 A first method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of drying a plant containing the ice crystallization inhibitor; and an ice crystallization inhibitor is extracted from the dried plant. Including the step of: Hereinafter, the method will be described for each step.
(1) 乾燥工程
本発明における乾燥工程により、植物の有する氷結晶化阻害物質の活性を著しく向上させることができる。植物から氷結晶化阻害物質を抽出する前に乾燥することでより優れた氷結晶化阻害活性を有する植物抽出液が得られるということは、これまで全く報告の無かった新たな知見である。(1) Drying process The activity of the ice crystallization inhibitory substance possessed by the plant can be significantly improved by the drying process in the present invention. It is a new finding that has never been reported so far that a plant extract having superior ice crystallization inhibitory activity can be obtained by drying before extracting an ice crystallization inhibitor from a plant.
本発明に係る氷結晶化阻害物質は、氷結晶の成長を阻害する機能を有する物質を意味するものであり、とくに限定されるものではないが、タンパク質を含む植物由来の抽出物であり、熱ヒステリシスの測定、氷結晶構造の観察、氷再結晶化阻害の測定など公知の方法により定義される氷結晶化阻害活性を有するものである。本発明に係る氷結晶化阻害物質は、単一の化合物であっても、複数の化合物の混合物であってもよい。 The ice crystallization inhibitor according to the present invention means a substance having a function of inhibiting the growth of ice crystals, and is not particularly limited, but is a plant-derived extract containing protein, It has ice crystallization inhibitory activity defined by a known method such as measurement of hysteresis, observation of ice crystal structure, measurement of ice recrystallization inhibition. The ice crystallization inhibitor according to the present invention may be a single compound or a mixture of a plurality of compounds.
本発明方法で用いる植物は、氷結晶化阻害物質を含む植物であれば特に限定されるものではないが、例えば、アブラナ科、セリ科、ユリ科またはキク科に属する植物が挙げられる。アブラナ科に属する植物は、ハクサイ、ダイコン、ブロッコリー、チンゲンサイ、コマツナ、カブ、シロナ、野沢菜、広島菜、ミズナ、マスタード(Brassica juncea)等が挙げられる。セリ科に属する植物はニンジン等が、ユリ科に属する植物はネギ等が、キク科に属する植物は春菊等が挙げられる。これらの中では、アブラナ科植物が好ましく、特にBrassica junceaが好ましい。また、これらの植物の類縁品種および改良品種も適宜使用することができる。 The plant used in the method of the present invention is not particularly limited as long as it contains an ice crystallization inhibitor, and examples thereof include plants belonging to the Brassicaceae, Aceraceae, Lilyaceae or Asteraceae families. Examples of plants belonging to the Brassicaceae include Chinese cabbage, Japanese radish, broccoli, Chingensai, Komatsuna, turnip, Sirona, Nozawana, Hiroshima rape, Mizuna, Mustard (Brassica juncea) and the like. Examples of the plant belonging to the family Aceraceae include carrots, examples of the plant belonging to the family Liliaceae include leeks, and examples of the plant belonging to the family Asteraceae include spring chrysanthemums. Among these, cruciferous plants are preferable, and Brassica juncea is particularly preferable. In addition, related varieties and improved varieties of these plants can also be used as appropriate.
乾燥に供する植物の形態は、特に限定されるものではなく、植物体の全体でもよく、例えば芽、葉、葉柄等、その一部分であってもよい。また、必要に応じて粉砕や細断等の加工を施したものであってもよい。さらに、後述するように低温馴化など公知の方法によって植物中の氷結晶化阻害物質を適宜誘導した後にこれを乾燥に供してもよい。 The form of the plant subjected to drying is not particularly limited, and may be the whole plant, for example, a part thereof such as a bud, a leaf, or a petiole. Moreover, what gave processing, such as a grinding | pulverization and shredding as needed, may be used. Furthermore, as described later, an ice crystallization inhibitor in a plant may be appropriately induced by a known method such as low-temperature acclimation, and then subjected to drying.
本発明に係る植物は、特に限定されるものではないが、好ましくはスプラウトの状態で使用する。本明細書において「スプラウト」とは、植物の新芽を意味するものである。例えば、ラディッシュ、ブロッコリー、マスタード(Brassica juncea)、クレス、レッドキャベツ、ケール等の新芽や、カイワレ大根やモヤシを好適に用いることができる。 The plant according to the present invention is not particularly limited, but is preferably used in a sprout state. In the present specification, “sprout” means plant sprout. For example, shoots such as radishes, broccoli, mustard (Brassica juncea), cress, red cabbage, kale, etc., radish radish and sprout can be suitably used.
本発明方法においては、乾燥工程の前に、植物を低温馴化する工程などにより植物中の氷結晶化物質を誘導してもよい。低温馴化の温度としては、特に限定されるものではないが、0℃以上、20℃以下が好ましい。また低温馴化の期間としては、特に限定されるものではないが、1日以上、より好ましくは3日間以上行うことが好ましい。低温馴化期間の上限は特に制限されないが、長過ぎると製造効率を損なうため、30日以下が好ましく、20日以下がより好ましく、15日以下がさらに好ましい。 In the method of the present invention, the ice crystallization substance in the plant may be induced by a step of acclimating the plant at a low temperature before the drying step. Although it does not specifically limit as temperature of low temperature acclimatization, 0 degreeC or more and 20 degrees C or less are preferable. The period of low temperature acclimation is not particularly limited, but it is preferably 1 day or longer, more preferably 3 days or longer. The upper limit of the low-temperature acclimation period is not particularly limited, but if it is too long, the production efficiency is impaired, so it is preferably 30 days or less, more preferably 20 days or less, and even more preferably 15 days or less.
乾燥処理の方法としては特に限定されるものではなく、凍結乾燥法、通風乾燥法、真空乾燥法を含む減圧乾燥法、蒸気乾燥法、高周波乾燥法、およびそれらを組合せた方法などを用いることができる。その中でも凍結乾燥法または通風乾燥法が好ましく、通風乾燥法としては特に温風乾燥または熱風乾燥が好ましい。 The drying method is not particularly limited, and it is possible to use a freeze-drying method, a ventilation drying method, a vacuum drying method including a vacuum drying method, a steam drying method, a high-frequency drying method, and a combination thereof. it can. Among them, the freeze drying method or the air drying method is preferable, and the air drying method is particularly preferably hot air drying or hot air drying.
乾燥処理の温度は、特に限定されるものではないが、−80℃以上、160℃以下の範囲で行うことが好ましい。具体的には、乾燥方法に応じて適宜調節することができる。例えば通風乾燥の温度条件としては、特に限定されるものではないが、植物中の氷結晶化物質の安定性や経済的な処理を実現する観点から、0℃以上が好ましく、4℃以上がより好ましく、10℃以上がさらに好ましく、15℃以上がさらに好ましく、最も好ましくは20℃以上であり、また、160℃以下が好ましく、150℃以下がより好ましく、140℃以下がさらに好ましく、130℃以下がさらに好ましく、最も好ましくは120℃以下である。 Although the temperature of a drying process is not specifically limited, It is preferable to carry out in -80 degreeC or more and 160 degrees C or less. Specifically, it can be appropriately adjusted according to the drying method. For example, the temperature condition for ventilation drying is not particularly limited, but is preferably 0 ° C. or higher and more preferably 4 ° C. or higher from the viewpoint of realizing the stability and economical treatment of the ice crystallization substance in the plant. Preferably, 10 ° C or higher is more preferable, 15 ° C or higher is further preferable, most preferably 20 ° C or higher, 160 ° C or lower is preferable, 150 ° C or lower is more preferable, 140 ° C or lower is further preferable, and 130 ° C or lower is preferable. Is more preferable, and most preferably 120 ° C. or lower.
乾燥時間は特に限定されず、用いる植物の種類や乾燥方法などに応じ、より一層優れた氷結晶化阻害活性を示す植物抽出物が得られる範囲で適宜調整すればよいが、通常、15分間以上、72時間以下とすることができ、30分間以上、48時間以下がより好ましく、1時間以上、24時間以下がさらに好ましい。 The drying time is not particularly limited, and may be appropriately adjusted within a range in which a plant extract exhibiting further excellent ice crystallization inhibitory activity is obtained depending on the type of plant used, the drying method, and the like. 72 hours or less, 30 minutes or more and 48 hours or less are more preferable, and 1 hour or more and 24 hours or less are more preferable.
本発明方法では、乾燥した植物をそのまま次の抽出工程に付してもよいが、乾燥後、粉砕や細断を行ってもよい。 In the method of the present invention, the dried plant may be subjected to the next extraction step as it is, but may be pulverized or shredded after drying.
(2) 抽出工程
次に、上記乾燥工程において植物内で活性が向上した氷結晶化阻害物質を抽出する。(2) Extraction step Next, an ice crystallization inhibitor having improved activity in the plant in the drying step is extracted.
乾燥処理後の植物から氷結晶化阻害物質を抽出する溶媒は特に限定されるものではないが、水、有機溶媒、超臨界二酸化炭素、亜臨界水などが好ましく例示される。有機溶媒としては、酢酸エチル、メタノール、エタノール、ヘキサン等が挙げられるが、食品加工に使用可能なものであることが好ましく、エタノール等が好ましい。これらの溶媒の中でも水またはエタノールが好ましく、水がより好ましい。また、水とエタノールとの混合溶媒など、水と有機溶媒を混合して用いることも可能である。なお、水には、酢酸ナトリウム緩衝液などの緩衝液が含まれるものとする。 The solvent for extracting the ice crystallization inhibitor from the plant after the drying treatment is not particularly limited, but water, organic solvent, supercritical carbon dioxide, subcritical water and the like are preferably exemplified. Examples of the organic solvent include ethyl acetate, methanol, ethanol, hexane, and the like, but those that can be used for food processing are preferable, and ethanol and the like are preferable. Among these solvents, water or ethanol is preferable, and water is more preferable. It is also possible to use a mixture of water and an organic solvent such as a mixed solvent of water and ethanol. The water includes a buffer solution such as a sodium acetate buffer solution.
水を用いる場合は、熱水を用いることが好ましく、有機溶媒を用いる場合は、加温した有機溶媒を用いることが好ましい。熱水又は加温した有機溶媒の温度として特に限定されないが、0℃以上、160℃以下が好ましく、20℃以上、120℃以下がより好ましい。抽出溶媒の種類や量は、抽出に処する植物体の種類や量により適宜選択することができる。 When using water, it is preferable to use hot water, and when using an organic solvent, it is preferable to use a heated organic solvent. Although it does not specifically limit as temperature of a hot water or the heated organic solvent, 0 to 160 degreeC is preferable and 20 to 120 degreeC is more preferable. The type and amount of the extraction solvent can be appropriately selected depending on the type and amount of the plant body subjected to extraction.
抽出物の形態は、抽出溶媒などにより異なる。例えば超臨界二酸化炭素を用いて得られた抽出物は固体として得られ、水や有機溶媒などを用いて得られた抽出物は液体として得られる。抽出液は、減圧濃縮などにより溶媒を除去して固体としてもよい。 The form of the extract varies depending on the extraction solvent and the like. For example, an extract obtained using supercritical carbon dioxide is obtained as a solid, and an extract obtained using water or an organic solvent is obtained as a liquid. The extract may be solid by removing the solvent by concentration under reduced pressure or the like.
(3) 吸着体処理工程
以上の工程を経て得られた氷結晶化阻害物質を含む抽出物は、さらに吸着体で処理してもよい。その詳しい条件などは、後述する第二方法で説明する。(3) Adsorbent treatment step The extract containing the ice crystallization inhibitor obtained through the above steps may be further treated with an adsorbent. The detailed conditions will be described in the second method described later.
本発明に係る氷結晶化阻害物質を含む植物抽出物を製造するための第二方法は、植物から氷結晶化阻害物質を抽出する工程;および、抽出物を吸着体で処理する工程を含むことを特徴とする。 A second method for producing a plant extract containing an ice crystallization inhibitor according to the present invention includes a step of extracting an ice crystallization inhibitor from a plant; and a step of treating the extract with an adsorbent. It is characterized by.
(1’) 抽出工程
本発明に係る第二方法では、植物から氷結晶化阻害物質を抽出する。その詳しい条件などは、第一方法の説明と同様のものとすることができる。また、抽出工程の前に、第一方法と同様に植物の乾燥工程を行ってもよいし、さらに乾燥工程前に低温馴化工程を行ってもよい。(1 ′) Extraction Step In the second method according to the present invention, an ice crystallization inhibitor is extracted from a plant. The detailed conditions can be the same as those described in the first method. Moreover, the drying process of a plant may be performed similarly to a 1st method before an extraction process, and also a low temperature acclimatization process may be performed before a drying process.
(2’) 吸着体処理工程
本発明に係る第二方法では、抽出工程で得られた抽出物と吸着体を接触させることにより、氷結晶化阻害物質以外の不純物の量を低減する。かかる吸着体処理により植物抽出液中の不要分を選択的に除去し、所要の氷結晶化阻害物質の精製度を著しく向上させることができる。さらに、除去される不要分には多くの植物抽出物が有する色素成分や香気成分なども含まれることから、植物抽出物の脱色・脱臭の効果も併せて期待できる。(2 ′) Adsorbent treatment step In the second method according to the present invention, the amount of impurities other than the ice crystallization inhibitor is reduced by bringing the extract obtained in the extraction step into contact with the adsorbent. By such an adsorbent treatment, unnecessary components in the plant extract can be selectively removed, and the required degree of purification of the ice crystallization inhibitor can be significantly improved. Furthermore, since the unnecessary components to be removed include pigment components, aroma components, and the like that many plant extracts have, the effects of decolorization and deodorization of plant extracts can also be expected.
抽出物は、液体であればそのまま吸着体と接触させてもよいし、或いは事前に濃縮または希釈してもよい。抽出物が固体である場合には、水、有機溶媒、水と有機溶媒との混合溶媒などに適宜溶解または分散する。 If the extract is liquid, it may be brought into contact with the adsorbent as it is, or may be concentrated or diluted in advance. When the extract is a solid, it is dissolved or dispersed as appropriate in water, an organic solvent, a mixed solvent of water and an organic solvent, or the like.
使用する吸着体は、特に限定されるものではないが、例えば合成吸着体、陽イオン交換樹脂、陰イオン交換樹脂、架橋デキストラン誘導体、ポリビニル系樹脂、アガロース誘導体、セルロース誘導体等の有機吸着体;活性炭、シリカゲル、アルミナ、ゼオライト、セピオライト、珪藻土、白土(活性白土、酸性白土)等の無機吸着体;親油性合成樹脂;綿花セルロース;再生綿;珪砂;海砂等が挙げられる。 The adsorbent used is not particularly limited. For example, an organic adsorbent such as a synthetic adsorbent, a cation exchange resin, an anion exchange resin, a crosslinked dextran derivative, a polyvinyl resin, an agarose derivative, or a cellulose derivative; activated carbon Inorganic adsorbents such as silica gel, alumina, zeolite, sepiolite, diatomaceous earth, white clay (active clay, acidic clay); lipophilic synthetic resin; cotton cellulose; regenerated cotton; quartz sand;
合成吸着体は、その材質により、芳香族系合成吸着体、置換芳香族系合成吸着体、アクリル系合成吸着体などに分類され、例えばセパビーズ(登録商標)SP850及びダイヤイオン(登録商標)HP20(以上、三菱化学株式会社製)、アンバーライト(登録商標)XAD−4、XAD−16、XAD−1180及びXAD−2000(以上、株式会社オルガノ製)等の芳香族系合成吸着体や、セパビーズSP205、SP206、SP207(以上、三菱化学株式会社製)等の置換芳香族系合成吸着体、ダイヤイオンHP2MG(三菱化学株式会社製)やアンバーライトXAD−7(株式会社オルガノ製)等のアクリル系合成吸着体等が挙げられる。 Synthetic adsorbents are classified into aromatic synthetic adsorbents, substituted aromatic synthetic adsorbents, acrylic synthetic adsorbents, and the like depending on the material. For example, Sepabeads (registered trademark) SP850 and Diaion (registered trademark) HP20 ( As described above, aromatic synthetic adsorbents such as Amberlite (registered trademark) XAD-4, XAD-16, XAD-1180, and XAD-2000 (manufactured by Organo Co., Ltd.), Sepabead SP205 , SP206, SP207 (Mitsubishi Chemical Co., Ltd.) substituted aromatic synthetic adsorbents, Diaion HP2MG (Mitsubishi Chemical Co., Ltd.) and Amberlite XAD-7 (Organo Co., Ltd.) acrylic synthesis Examples include adsorbents.
陽イオン交換樹脂からなる吸着体としては、例えばスルホン酸イオンをイオン交換基とする樹脂である、アンバーライトCG−4000、CG−5000、CG−6000、CG−8000、IR−116、IR−118、IR−120B、IR−122、IR−124、XT−1007、XT−1009、XT−1002(以上、株式会社オルガノ製)や、アクリル酸系又はメタアクリル酸系の樹脂である、ダイヤイオンWK10、WK20(以上、三菱化学株式会社製)などが挙げられる。 As an adsorbent comprising a cation exchange resin, for example, Amberlite CG-4000, CG-5000, CG-6000, CG-8000, IR-116, IR-118, which are resins having sulfonate ions as ion exchange groups. , IR-120B, IR-122, IR-124, XT-1007, XT-1009, XT-1002 (manufactured by Organo Co., Ltd.) and Diaion WK10, which is an acrylic acid or methacrylic acid resin And WK20 (manufactured by Mitsubishi Chemical Corporation).
陰イオン交換樹脂からなる吸着体としては、例えば2〜3級アミノ基又は第4級アンモニウム基をイオン交換基とする樹脂であるダイヤイオンWA10、WA20、WA30、WA21J、SA20A(以上、三菱化学株式会社製)などが挙げられる。 Examples of the adsorbent made of anion exchange resin include Diaion WA10, WA20, WA30, WA21J, SA20A (Mitsubishi Chemical Co., Ltd.), which are resins having a secondary or tertiary amino group or a quaternary ammonium group as an ion exchange group. Company-made).
架橋デキストラン誘導体からなる吸着体としては、例えばセファデックス(登録商標)LH20、LH60(以上、GEヘルスケアバイオサイエンス株式会社製)などが挙げられる。 Examples of the adsorbent made of a crosslinked dextran derivative include Sephadex (registered trademark) LH20 and LH60 (manufactured by GE Healthcare Biosciences, Inc.).
ポリビニル系樹脂からなる吸着体としては、例えばトヨパール(登録商標)HW−40、50(東ソー株式会社製)などが挙げられる。 Examples of the adsorbent made of polyvinyl resin include Toyopearl (registered trademark) HW-40, 50 (manufactured by Tosoh Corporation).
アガロース誘導体からなる吸着体としては、例えばセファロース(登録商標)CL、4B、6B(以上GEヘルスケアバイオサイエンス株式会社製)などが挙げられる。 Examples of the adsorbent composed of an agarose derivative include Sepharose (registered trademark) CL, 4B, and 6B (manufactured by GE Healthcare Bioscience Co., Ltd.).
セルロース誘導体からなる吸着体としては、例えばセルロファイン(登録商標)CL−90、GCL−300、GCL−1000(以上、生化学工業株式会社製)などが挙げられる。 Examples of the adsorbent composed of a cellulose derivative include Cellulofine (registered trademark) CL-90, GCL-300, GCL-1000 (manufactured by Seikagaku Corporation) and the like.
活性炭は、その形状から、粉末状活性炭、粒状活性炭、破砕状炭、球形炭、繊維状活性炭等に分類される。その由来原料としては、例えばオガコ、木炭、椰子殻等の植物原料、石炭等の化石原料、フェノール樹脂、ポリエステル樹脂等の合成樹脂原料などが挙げられ、これらの原料を炭化、賦活して各種の活性炭が製造される。賦活には例えば水蒸気や塩化水素、一酸化炭素、二酸化炭素、酸素等を用いるガス賦活;アルカリ、酸又は塩による薬品賦活等が挙げられる。本発明に用いられる活性炭としては、例えば、粉末状活性炭である太閤S、太閤FC、太閤K(以上、二村化学工業株式会社製)や、活性炭素粉末(和光純薬工業製)等を用いることができる。 Activated carbon is classified according to its shape into powdered activated carbon, granular activated carbon, crushed coal, spherical charcoal, fibrous activated carbon, and the like. Examples of the raw materials include plant raw materials such as sawdust, charcoal, and coconut shells, fossil raw materials such as coal, synthetic resin raw materials such as phenol resins and polyester resins, and the like. Activated carbon is produced. Examples of the activation include gas activation using water vapor, hydrogen chloride, carbon monoxide, carbon dioxide, oxygen, etc .; chemical activation using an alkali, acid, or salt. As the activated carbon used in the present invention, for example, powdered activated carbons such as Dazai S, Dazai FC, Dazai K (above, manufactured by Nimura Chemical Co., Ltd.), activated carbon powder (manufactured by Wako Pure Chemical Industries, Ltd.) and the like are used. Can do.
シリカゲルとしては、公知のもの及び通常市販されているものをいずれも使用できる。形状も種々知られているが、とくに制限はない。例えばワコーゲルC−100、C−200、C−300(以上、和光純薬工業株式会社製)等を用いることができる。 As the silica gel, any known and usually commercially available products can be used. Various shapes are also known, but there is no particular limitation. For example, Wakogel C-100, C-200, C-300 (above, manufactured by Wako Pure Chemical Industries, Ltd.) and the like can be used.
ゼオライトは結晶中に微細孔を持つアルミノ珪酸塩の総称であり、例えばX型ゼオライト、Y型ゼオライト、モルデナイト等の天然ゼオライトや、A型ゼオライト、ホージャサイト型ゼオライト、ソーダライト型ゼオライトなどの合成ゼオライトが挙げられる。ゼオライトとしては、公知のもの及び通常市販されているものをいずれも使用できる。 Zeolite is a generic term for aluminosilicates with fine pores in the crystal. For example, natural zeolite such as X-type zeolite, Y-type zeolite, mordenite, synthetic zeolite such as A-type zeolite, faujasite-type zeolite, sodalite-type zeolite, etc. Is mentioned. As the zeolite, both known and usually commercially available ones can be used.
セピオライトは含水マグネシウム珪酸塩を主成分とする鎖状粘土鉱物であり、その結晶中にトンネルを持つ特異な鎖状結晶構造により吸着及び脱臭能を有する。セピオライトとしては、公知のもの及び通常市販されているものをいずれも使用できる。 Sepiolite is a chain clay mineral composed mainly of hydrous magnesium silicate, and has adsorption and deodorizing ability due to a unique chain crystal structure with a tunnel in the crystal. As sepiolite, both known and usually commercially available products can be used.
珪藻土は藻類の一種である珪藻の化石よりなる堆積物であり、二酸化ケイ素を主成分とする多孔体である。珪藻土としては、公知のもの及び通常市販されているものをいずれも使用できる。 Diatomaceous earth is a deposit made of fossil diatom, which is a kind of algae, and is a porous body mainly composed of silicon dioxide. As a diatomaceous earth, both a well-known thing and the normally marketed thing can be used.
活性白土や酸性白土などの白土は、シリカ層、アルミナ層、シリカ層の三層からなる結晶構造体であり、陽イオン交換能や吸着能を有している。白土としては、公知のもの及び通常市販されているものをいずれも使用できる。 White clay such as activated clay and acidic clay is a crystal structure composed of three layers of a silica layer, an alumina layer, and a silica layer, and has cation exchange capacity and adsorption capacity. As the clay, both publicly known and normally marketed ones can be used.
親油性合成樹脂としては、例えばポリエチレンやポリプロピレン、ポリエステル、ポリウレタン、ポリ塩化ビニル、ポリアクリル、ポリカーボネート等、公知のもの及び通常市販されているものをいずれも使用できる。 As the oleophilic synthetic resin, any of publicly known and usually commercially available ones such as polyethylene, polypropylene, polyester, polyurethane, polyvinyl chloride, polyacryl and polycarbonate can be used.
綿花セルロース、再生綿、珪砂、海砂は、いずれも公知のもの及び通常市販されているものをいずれも使用できる。 As the cotton cellulose, the regenerated cotton, the silica sand, and the sea sand, any known one and those that are usually marketed can be used.
上記の吸着体は、抽出液中の氷結晶化阻害物質との親和性が低く、且つ抽出液中の他の成分との吸着性が高いという性質を有することが好ましい。そのような吸着体としては、例えば、活性炭が好ましい。抽出液との混合等、操作性の観点から、粉末状活性炭が特に好ましい。 The adsorbent preferably has a property of low affinity with an ice crystallization inhibitor in the extract and high adsorbability with other components in the extract. As such an adsorbent, for example, activated carbon is preferable. From the viewpoint of operability such as mixing with the extract, powdered activated carbon is particularly preferable.
吸着体の添加量は、植物抽出液に含まれる固形分量、タンパク量、溶媒の種類、吸着体の種類等に応じて適宜設定すればよい。特に限定されるものではないが、例えば、抽出物中のタンパク質1重量部に対して0.001重量部以上が好ましく、0.01重量部以上がより好ましく、0.1重量部以上がさらに好ましく、また、100重量部以下が好ましく、80重量部以下がより好ましく、50重量部以下がさらに好ましい。上記割合が0.001重量部よりも少ないと不要分の除去が不充分である可能性があり、100重量部を超えると処理後の溶液の回収率が悪くなるという問題が生じ得るため、好ましくない。 What is necessary is just to set the addition amount of an adsorbent suitably according to the solid content amount contained in a plant extract, the amount of protein, the kind of solvent, the kind of adsorbent, etc. Although not particularly limited, for example, 0.001 part by weight or more is preferable with respect to 1 part by weight of protein in the extract, 0.01 part by weight or more is more preferable, and 0.1 part by weight or more is more preferable. Moreover, 100 weight part or less is preferable, 80 weight part or less is more preferable, and 50 weight part or less is further more preferable. If the above ratio is less than 0.001 part by weight, the unnecessary part may be insufficiently removed, and if it exceeds 100 parts by weight, there may be a problem that the recovery rate of the solution after treatment may be deteriorated. Absent.
抽出物と吸着体との接触は、いかなる方法で行ってもよい。例えば、吸着体を抽出物に加え、一定時間後に吸着体を除去するバッチ法や、吸着体をカラムに充填し、これに抽出物を通過させるカラム法などが挙げられる。なお、上述したように、抽出物が液体であればそのまま吸着体と接触させればよいし、固体であれば溶液または分散液とした上で接触させればよい。 The contact between the extract and the adsorbent may be performed by any method. For example, a batch method in which an adsorbent is added to an extract and the adsorbent is removed after a predetermined time, a column method in which an adsorbent is packed in a column, and an extract is passed through the column can be used. As described above, if the extract is a liquid, it may be brought into contact with the adsorbent as it is. If it is a solid, it may be brought into contact with the solution or dispersion.
抽出物と吸着体との接触時間や吸着処理の回数などは、抽出物に含まれるタンパク質や抽出物の氷結晶化阻害活性などに応じて適宜調節すればよい。 What is necessary is just to adjust suitably the contact time of an extract and an adsorbent, the frequency | count of adsorption processing, etc. according to the protein contained in an extract, the ice crystallization inhibitory activity of an extract, etc.
以上で説明した本発明に係る方法により製造された植物抽出物は、氷結晶化阻害活性を有する。 The plant extract produced by the method according to the present invention described above has ice crystallization inhibitory activity.
本発明に係る氷結晶化阻害活性を有する植物抽出物は、水が氷結晶化することで障害が生じる様々な分野において、この障害を抑制する目的で利用可能である。例えば、食品分野、機械分野、土木分野、化粧品分野、生体材料を用いる医療分野等で利用可能である。 The plant extract which has the ice crystallization inhibitory activity which concerns on this invention can be utilized in order to suppress this disorder | damage | failure in the various field | areas where a disorder | damage | failure arises because water crystallizes ice. For example, it can be used in the food field, machine field, civil engineering field, cosmetics field, medical field using biomaterials, and the like.
食品分野では、食品に含まれる水の氷結晶化を抑制することで、当該食品の味の劣化等を防ぐことができる。例えば、澱粉老化を防止したり、また、食品中の水が氷結晶化して、タンパク質や油脂成分等を物理的に圧迫して、その構造を変化させることによる味や品質等の劣化を抑制したりすることができる。 In the food field, by suppressing the ice crystallization of water contained in the food, deterioration of the taste of the food can be prevented. For example, starch aging is prevented, and water in food is crystallized in ice, and protein, fat and oil components, etc. are physically pressed to suppress deterioration of taste, quality, etc. caused by changing its structure. Can be.
機械分野、土木分野では、機械の可動部、道路、地盤等の凍結防止剤として利用できる。 In the mechanical field and civil engineering field, it can be used as an antifreezing agent for moving parts, roads, ground, etc. of machines.
化粧品分野では、化粧品の品質の劣化等を防ぐための添加剤として利用できる。例えば、油脂成分を含む化粧品を凍結保存のため凍結させると、当該化粧品に含まれる水が氷結晶化して、当該油脂成分を物理的に圧迫してその構造を壊すことがあり、品質及び使用感が劣化するため、従来、化粧品の凍結保存は困難であった。しかし本発明に係る植物抽出物を用いれば、水の氷結晶化を防ぐことで油脂成分の構造が保持されるため、品質の劣化等を抑制することができる。 In the cosmetic field, it can be used as an additive for preventing deterioration of cosmetic quality. For example, if a cosmetic containing an oil / fat component is frozen for cryopreservation, the water contained in the cosmetic may crystallize in ice and physically compress the oil / fat component to break its structure. Conventionally, it has been difficult to freeze and store cosmetics. However, if the plant extract according to the present invention is used, the structure of the oil and fat component is retained by preventing ice crystallization of water, so that deterioration of quality and the like can be suppressed.
医療分野では、生体材料を凍結保存する際の保護剤として用いることができる。例えば、細胞、血液、臓器等の組織等の生体材料を従来公知の保存液に入れて凍結保存すると、保存液中の水分が凍結して氷結晶を生じ、当該氷結晶により生体材料が損傷することがある。しかし、本発明に係る植物抽出物を添加すれば、氷結晶の発生、成長を抑制することができるので、生体材料を氷結晶による損傷から保護することができる。 In the medical field, it can be used as a protective agent when cryopreserving a biomaterial. For example, when biomaterials such as cells, blood, organs, etc. are placed in a conventionally known preservation solution and frozen and stored, the water in the preservation solution freezes to produce ice crystals, which damage the biological materials. Sometimes. However, if the plant extract according to the present invention is added, the generation and growth of ice crystals can be suppressed, so that the biomaterial can be protected from damage due to ice crystals.
本発明の植物抽出物はそのまま用いることができるが、必要に応じて、さらに精製を行ってもよい。例えば、デカンテーション、濾過、遠心分離等により夾雑成分を除去してもよい。また例えば、塩析や有機溶媒による沈殿や、アフィニティークロマトグラフィー、イオン交換カラムクロマトグラフィー、ゲル濾過等による精製、透析や限外濾過等による濃縮等を行ってもよい。さらに必要により、粉末状または顆粒状など任意の形態に固形化してもよい。固形化の方法はとくに限定されないが、例えば、上記の抽出物を噴霧乾燥や凍結乾燥等の常法に従って粉末化する方法や、抽出物を賦形剤に吸着、担持させて粉末または顆粒状に固形化する方法などを挙げることができる。これらの操作は当業者に公知のものであり、用途に応じて適宜選択して用いることができる。 Although the plant extract of this invention can be used as it is, you may refine | purify further as needed. For example, contaminant components may be removed by decantation, filtration, centrifugation, or the like. Further, for example, salting out, precipitation with an organic solvent, purification by affinity chromatography, ion exchange column chromatography, gel filtration, etc., concentration by dialysis, ultrafiltration, or the like may be performed. Further, if necessary, it may be solidified into an arbitrary form such as powder or granule. The solidification method is not particularly limited. For example, the above-described extract is pulverized according to a conventional method such as spray drying or freeze-drying, or the extract is adsorbed and supported on an excipient to form powder or granules. Examples of the method include solidification. These operations are known to those skilled in the art, and can be appropriately selected and used according to the application.
本発明の植物抽出物は、そのまま用いてもよいし、或いは、植物抽出物または植物抽出物の乾燥物を含む氷結晶化阻害物質含有組成物として用いてもよい。その形態は、用途に応じて様々であり、例えば、溶液、濃縮液、懸濁液、凍結乾燥物、粉末、顆粒、錠剤などであってもよい。 The plant extract of the present invention may be used as it is, or may be used as a composition containing an ice crystallization inhibitor containing a plant extract or a dried product of the plant extract. The form varies depending on the application, and may be, for example, a solution, a concentrate, a suspension, a lyophilized product, a powder, a granule, a tablet, or the like.
次に、本発明に係る植物抽出物の氷結晶化阻害活性の測定方法、及びタンパク質量の測定方法について説明する。 Next, the measuring method of the ice crystallization inhibitory activity of the plant extract which concerns on this invention, and the measuring method of protein amount are demonstrated.
本発明の抽出物の氷結晶化阻害活性の測定方法は、植物の種類などに応じて適宜適したものを用いる。例えば、熱ヒステリシスの測定、氷結晶構造の観察、氷再結晶化阻害の測定などの公知の方法にて行うことができ、何れかの方法で氷結晶化阻害活性の向上が認められる場合は、本発明範囲に含まれるものとする。氷結晶化阻害活性の測定は、例えば、ショ糖を30w/v%含む植物抽出物溶液を−40℃に冷却した後に−6℃まで温度を上げ、顕微鏡により観察した氷結晶の平均面積を測定することにより行うことができる。氷結晶化阻害活性が強いほどこの氷結晶の平均面積は小さくなることから、この数値を指標として、植物抽出物の氷結晶化阻害活性を定量的に評価することができる。なお、ここで測定できる「植物抽出物の氷結晶化阻害活性」とは、植物抽出物から溶媒を除去した固形分に水とショ糖を加えて30w/v%のショ糖を含む溶液とした場合における氷結晶化阻害活性をいうものとする。 As the method for measuring the ice crystallization inhibitory activity of the extract of the present invention, an appropriate method is used according to the type of plant. For example, it can be performed by a known method such as measurement of thermal hysteresis, observation of ice crystal structure, measurement of ice recrystallization inhibition, etc., and if any method shows improvement in ice crystallization inhibition activity, It is intended to be included in the scope of the present invention. The ice crystallization inhibitory activity is measured, for example, by cooling the plant extract solution containing 30 w / v% sucrose to −40 ° C., then raising the temperature to −6 ° C., and measuring the average area of ice crystals observed with a microscope This can be done. The stronger the ice crystallization inhibitory activity is, the smaller the average area of the ice crystals is. Therefore, the ice crystallization inhibitory activity of the plant extract can be quantitatively evaluated using this value as an index. The “ice crystallization inhibitory activity of plant extract” that can be measured here is a solution containing 30 w / v% sucrose by adding water and sucrose to the solid content obtained by removing the solvent from the plant extract. In this case, it means the ice crystallization inhibitory activity.
本発明に係る第一方法により得られた植物抽出物の氷結晶化阻害活性は、例えば、上記方法で測定された氷結晶の平均面積を、乾燥工程を経ない以外は同様の製造方法で得られる植物抽出物を同様に処理して測定される平均面積に比べることにより測定することができる。本発明に係る第一方法により得られた植物抽出物としては、上記方法で測定された氷結晶の平均面積が、乾燥工程を経ない以外は同様の製造方法で得られる植物抽出物を同様に処理して測定される平均面積に比べて5%以上低いものが好ましい。 The ice crystallization inhibitory activity of the plant extract obtained by the first method according to the present invention is obtained, for example, by the same production method except that the average area of ice crystals measured by the above method is not subjected to a drying step. It can be measured by comparing the average area measured by treating the plant extract obtained in the same manner. The plant extract obtained by the first method according to the present invention is the same as the plant extract obtained by the same production method except that the average area of ice crystals measured by the above method does not go through the drying step. What is 5% or more lower than the average area measured by processing is preferable.
本発明の抽出物のタンパク質量の測定方法は、特に限定されるものではないが、例えばLowry法やビシンコニン酸(BCA)法、Bradford法(Coomassie法)などの公知の方法にて行うことができる。標準タンパク質としては、特に限定されないが、例えばウシ血清アルブミン(BSA)を好適に用いることができる。 The method for measuring the protein amount of the extract of the present invention is not particularly limited, and can be performed by a known method such as the Lowry method, the bicinchoninic acid (BCA) method, the Bradford method (Coomassie method), or the like. . Although it does not specifically limit as a standard protein, For example, bovine serum albumin (BSA) can be used suitably.
以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の範囲に含まれる。また、本明細書中に記載された特許文献及び非特許文献の全てが、本明細書中において参考として援用される。 Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the scope of the invention. Moreover, all the patent documents and nonpatent literatures described in this specification are used as reference in this specification.
実施例1 乾燥後抽出による抽出物の製造
市販のマスタードスプラウト(村上農園製,40g)を−80℃の冷凍庫内で一晩凍結した後に、凍結乾燥機(東京理化機器製,品番:FD−5N)で乾燥し、乾燥物(2.0g)を得た。この乾燥物に水(80mL)を加え、105℃、0.12MPaで20分間加圧抽出した。濾紙(アドバンテック社製,保留粒子径:5μm)を用いて濾過した後、回収した濾液を減圧濃縮装置(東京理化機器製ロータリーエバポレーター,品番:N−1000−S−W)で減圧乾燥し、抽出物(820mg)を得た。さらにこの抽出物を濃度が100mg/mLとなるように水に溶解した。この溶液のタンパク質濃度を、ピアース社製のBCAキットを用い、BCA法によって測定したところ、27.8mg/mLであった。Example 1 Manufacture of an extract by extraction after drying A commercial mustard sprout (Murakami Farm, 40 g) was frozen overnight in a freezer at −80 ° C., and then freeze-dried (Tokyo Rika Kikai Co., Ltd., product number: FD-5N). ) To obtain a dried product (2.0 g). Water (80 mL) was added to the dried product, and pressure extraction was performed at 105 ° C. and 0.12 MPa for 20 minutes. After filtration using a filter paper (manufactured by Advantech Co., Ltd., retained particle size: 5 μm), the recovered filtrate is dried under reduced pressure with a vacuum concentrator (Tokyo Rika Co., Ltd. rotary evaporator, product number: N-1000-SW) and extracted. Product (820 mg) was obtained. Further, this extract was dissolved in water so as to have a concentration of 100 mg / mL. The protein concentration of this solution was measured by the BCA method using a Pierce BCA kit and found to be 27.8 mg / mL.
実施例2 乾燥後抽出による抽出物の製造
実施例1に記載のマスタードスプラウト(40g)を金属製トレーにとり、温度40℃の恒温乾燥機内にて温風乾燥し、乾燥物(1.8g)を得た。実施例1に記載の方法と同様に、この乾燥物を熱水抽出した後に濾液を濃縮し、得られた抽出物(920mg)を濃度が100mg/mLとなるように水に溶解した。この溶液のタンパク質濃度をBCA法によって測定したところ、25.4mg/mLであった。Example 2 Manufacture of extract by extraction after drying The mustard sprout (40 g) described in Example 1 was placed in a metal tray and dried in warm air in a constant temperature dryer at a temperature of 40 ° C. to obtain a dried product (1.8 g). Obtained. In the same manner as described in Example 1, this dried product was extracted with hot water, and then the filtrate was concentrated. The obtained extract (920 mg) was dissolved in water to a concentration of 100 mg / mL. When the protein concentration of this solution was measured by the BCA method, it was 25.4 mg / mL.
比較例1
実施例1に記載のマスタードスプラウト(40g)を乾燥せず、そのまま水(80mL)を加えた以外は実施例1と同様にして、105℃、0.12MPaで20分間加圧抽出し、抽出物(860mg)を得た。この抽出物を濃度が100mg/mLとなるよう水に溶解し、溶液のタンパク質濃度をBCA法で測定したところ、30.1mg/mLであった。Comparative Example 1
The mustard sprout (40 g) described in Example 1 was not dried and was subjected to pressure extraction at 105 ° C. and 0.12 MPa for 20 minutes in the same manner as in Example 1 except that water (80 mL) was added as it was. (860 mg) was obtained. This extract was dissolved in water to a concentration of 100 mg / mL, and the protein concentration of the solution was measured by the BCA method to be 30.1 mg / mL.
実施例3 氷結晶化阻害活性の測定
実施例1、2および比較例1で得た抽出物の水溶液を、60w/v%のショ糖溶液と1:1(v/v)の割合で混合した。当該混合物(1μL)をカバーガラスで挟み込んだ。加熱冷却載物台(LINKAM社製,LK‐600PM)を備えた光学顕微鏡(OLYMPUS社製,BX50)のガラスシャーレを20℃に保ち、その上に上記カバーガラスを載せて、100℃/minの速度で−40℃まで冷却した。次に、100℃/minの速度で−6℃まで温度を上げた。−6℃になった時を0分として、その後、光学顕微鏡をそのままの状態に保ち、30分後の画像を取り込んだ。得られた画像中に存在する氷結晶の平均面積を算出し、これを氷結晶化阻害活性の指標とした。この結果を表1に示す。表1は、それぞれの水溶液について測定して得られた画像中の氷結晶の平均面積を、比較例1の平均面積に対する比として数値化した結果である。Example 3 Measurement of ice crystallization inhibitory activity The aqueous solutions of the extracts obtained in Examples 1 and 2 and Comparative Example 1 were mixed with a 60 w / v% sucrose solution at a ratio of 1: 1 (v / v). . The mixture (1 μL) was sandwiched between cover glasses. Keep the glass petri dish of an optical microscope (OLYMPUS, BX50) equipped with a heating / cooling stage (LINKAM, LK-600PM) at 20 ° C., and place the cover glass on top of the glass dish. Cooled to -40 ° C at a rate. Next, the temperature was increased to −6 ° C. at a rate of 100 ° C./min. The time when the temperature reached −6 ° C. was set to 0 minute, and then the optical microscope was kept as it was, and the image after 30 minutes was captured. The average area of ice crystals present in the obtained image was calculated and used as an index of ice crystallization inhibitory activity. The results are shown in Table 1. Table 1 shows the results of quantifying the average area of ice crystals in the image obtained by measuring each aqueous solution as a ratio to the average area of Comparative Example 1.
表1に示すように、マスタードスプラウトを乾燥した後に抽出して得られた実施例1及び実施例2の抽出物の水溶液について氷結晶化阻害活性を測定した結果、氷結晶の平均面積は比較例1の抽出物の水溶液よりも小さい値であった。氷結晶の平均面積が小さいほど、氷結晶化阻害活性が強いことを示している。この結果から、凍結乾燥および40℃の温風乾燥によりマスタードスプラウトから抽出される氷結晶化阻害物質の氷結晶化阻害活性が向上することは明らかである。 As shown in Table 1, as a result of measuring the ice crystallization inhibitory activity of the aqueous solutions of the extracts of Example 1 and Example 2 obtained by extracting the mustard sprout after drying, the average area of the ice crystals is a comparative example. It was a value smaller than the aqueous solution of 1 extract. The smaller the average area of ice crystals, the stronger the ice crystallization inhibitory activity. From this result, it is clear that the ice crystallization inhibitory activity of the ice crystallization inhibitor extracted from mustard sprout is improved by freeze drying and hot air drying at 40 ° C.
実施例4 低温馴化と乾燥後の抽出による抽出物の製造
市販のマスタードスプラウトを15℃にて低温馴化し、氷結晶化阻害物質を誘導した。馴化10日目のマスタードスプラウト(25g)を実施例1に記載の方法と同様にして凍結乾燥し、乾燥物(1.3g)を得た。この乾燥物に水(50mL)を加え、105℃、0.12MPaで20分間加圧抽出した後に濾液を乾燥し、抽出物(330mg)を得た。この抽出物を濃度が100mg/mLとなるように溶解し、溶液のタンパク質濃度をBCA法によって測定したところ、26.6mg/mLであった。Example 4 Preparation of extract by low temperature acclimation and extraction after drying Commercial mustard sprout was conditioned at 15 ° C. to induce ice crystallization inhibitors. The mustard sprout of acclimatization day 10 (25 g) was freeze-dried in the same manner as described in Example 1 to obtain a dried product (1.3 g). Water (50 mL) was added to the dried product, and after pressure extraction at 105 ° C. and 0.12 MPa for 20 minutes, the filtrate was dried to obtain an extract (330 mg). This extract was dissolved to a concentration of 100 mg / mL, and the protein concentration of the solution was measured by the BCA method to be 26.6 mg / mL.
実施例5 低温馴化と乾燥後の抽出による抽出物の製造
実施例4で低温馴化したマスタードスプラウト(25g)を金属製トレーに取り、実施例2に記載の方法と同様にして40℃で温風乾燥し、乾燥物(1.2g)を得た。この乾燥物に水(50mL)を加え、105℃、0.12MPaで20分間加圧抽出した後に濾液を乾燥し、抽出物(240mg)を得た。この抽出物を濃度が100mg/mLとなるように溶解し、溶液のタンパク質濃度をBCA法によって測定したところ、30.1mg/mLであった。Example 5 Manufacture of extract by low temperature acclimation and extraction after drying The mustard sprout (25 g) conditioned at low temperature in Example 4 was placed in a metal tray and heated at 40 ° C. in the same manner as described in Example 2. Dried to obtain a dried product (1.2 g). Water (50 mL) was added to the dried product, followed by pressure extraction at 105 ° C. and 0.12 MPa for 20 minutes, and then the filtrate was dried to obtain an extract (240 mg). This extract was dissolved to a concentration of 100 mg / mL, and the protein concentration of the solution was measured by the BCA method to be 30.1 mg / mL.
実施例6 低温馴化と乾燥後の抽出による抽出物の製造
実施例4で低温馴化したマスタードスプラウト(25g)を金属製トレーに取り、温度90℃の恒温乾燥機内にて熱風乾燥し、乾燥物(2.3g)を得た。この乾燥物に水(50mL)を加え、105℃、0.12MPaで20分間加圧抽出した後に濾液を乾燥し、抽出物(300mg)を得た。この抽出物を濃度が100mg/mLとなるように溶解し、溶液のタンパク質濃度をBCA法によって測定したところ、22.5mg/mLであった。Example 6 Manufacture of extract by low temperature acclimation and extraction after drying The mustard sprout (25 g) conditioned in low temperature in Example 4 was placed in a metal tray and dried in hot air in a constant temperature dryer at 90 ° C. 2.3 g) was obtained. Water (50 mL) was added to the dried product, followed by pressure extraction at 105 ° C. and 0.12 MPa for 20 minutes, and then the filtrate was dried to obtain an extract (300 mg). This extract was dissolved to a concentration of 100 mg / mL, and the protein concentration of the solution was measured by the BCA method to be 22.5 mg / mL.
比較例2
実施例4で低温馴化したマスタードスプラウト(25g)を乾燥せず、そのまま水(50mL)を加え、105℃、0.12MPaで20分間加圧抽出し、抽出物(350mg)を得た。この抽出物を濃度が100mg/mLとなるように水に溶解し、溶液のタンパク質濃度をBCA法で測定したところ、24.6mg/mLであった。Comparative Example 2
The mustard sprout (25 g) conditioned at low temperature in Example 4 was not dried, water (50 mL) was added as it was, and pressure extraction was performed at 105 ° C. and 0.12 MPa for 20 minutes to obtain an extract (350 mg). This extract was dissolved in water to a concentration of 100 mg / mL, and the protein concentration of the solution was measured by the BCA method. As a result, it was 24.6 mg / mL.
実施例7 氷結晶化阻害活性の測定
実施例4、5、6および比較例2で得た抽出物の水溶液について、それぞれ実施例3に記載の方法と同様に氷結晶化阻害活性を測定した。得られた画像中の氷結晶の平均面積を比較例2の抽出物の水溶液について得られた氷結晶の平均面積に対する比として数値化した。この結果を表2に示す。Example 7 Measurement of Ice Crystallization Inhibitory Activity For the aqueous solutions of the extracts obtained in Examples 4, 5, 6 and Comparative Example 2, ice crystallization inhibitory activity was measured in the same manner as described in Example 3. The average area of ice crystals in the obtained image was quantified as a ratio to the average area of ice crystals obtained for the aqueous solution of the extract of Comparative Example 2. The results are shown in Table 2.
表2に示すように、マスタードスプラウトを低温馴化した後に乾燥した場合においても、乾燥後に抽出して得られる抽出液の水溶液の氷結晶の平均面積(実施例4ないし6)は、乾燥処理を施していない比較例2の抽出物の水溶液の氷結晶の平均面積よりも小さくなっていた。この結果から、低温馴化により氷結晶化阻害活性を誘導した後に、これを凍結乾燥、温風乾燥、または、熱風乾燥することにより、マスタードスプラウト中の氷結晶化物質の氷結晶化阻害活性が向上することは明らかである。 As shown in Table 2, even when the mustard sprout was dried after acclimation at low temperature, the average area of ice crystals in the aqueous solution of the extract obtained by extraction after drying (Examples 4 to 6) was subjected to a drying treatment. It was smaller than the average area of ice crystals of the aqueous solution of the extract of Comparative Example 2 that was not. From this result, after inducing ice crystallization inhibitory activity by acclimation at low temperature, freeze-drying, hot-air drying, or hot-air drying of this improves the ice crystallization inhibitory activity of the ice crystallization substance in mustard sprout. It is clear to do.
実施例8 低温馴化と乾燥後の抽出による抽出物の製造
市販のマスタードスプラウトを15℃にて低温馴化し、氷結晶化阻害物質を誘導した。馴化10日目のマスタードスプラウト(50g)を金属製トレーに取り、実施例2に記載の方法と同様にして40℃で温風乾燥し、乾燥物(4.1g)を得た。この乾燥物に水(100mL)を加え、50℃で2時間加熱抽出した後に濾液を乾燥し、抽出物(800mg)を得た。この抽出物のタンパク質濃度がBCA法により10mg/mLとなるように溶解し、抽出液(22mL)を得た。Example 8 Manufacture of extract by low temperature acclimation and extraction after drying A commercial mustard sprout was cold conditioned at 15 ° C. to induce ice crystallization inhibitors. 10 days of acclimatization mustard sprout (50 g) was placed in a metal tray and dried in hot air at 40 ° C. in the same manner as described in Example 2 to obtain a dried product (4.1 g). Water (100 mL) was added to the dried product, followed by heating and extraction at 50 ° C. for 2 hours, and then the filtrate was dried to obtain an extract (800 mg). The extract was dissolved so that the protein concentration was 10 mg / mL by the BCA method to obtain an extract (22 mL).
比較例3
実施例8で低温馴化したマスタードスプラウト(50g)を乾燥せず、そのまま水(100mL)を加え、50℃で2時間加熱抽出し、抽出物(880mg)を得た。この抽出物のタンパク質濃度がBCA法で10mg/mLとなるように溶解し、抽出液(26mL)を得た。Comparative Example 3
The mustard sprout (50 g) conditioned at low temperature in Example 8 was not dried, water (100 mL) was added as it was, and the mixture was extracted by heating at 50 ° C. for 2 hours to obtain an extract (880 mg). The extract was dissolved so that the protein concentration was 10 mg / mL by the BCA method to obtain an extract (26 mL).
実施例9 氷結晶化阻害活性の測定
実施例8および比較例3で得た抽出液について、それぞれ実施例3に記載の方法と同様に氷結晶化阻害活性を測定した。得られた画像中の氷結晶の平均面積を比較例3の抽出物の水溶液について得られた氷結晶の平均面積に対する比として数値化した結果、その値は0.88であり、乾燥後に抽出して得られる抽出液の水溶液の氷結晶の平均面積(実施例8)は、乾燥処理を施していない比較例3の抽出物の水溶液の氷結晶の平均面積よりも小さくなっていた。この結果からも、低温馴化による氷結晶化阻害活性の誘導後にこれを乾燥することによって、マスタードスプラウト中の氷結晶化物質の氷結晶化阻害活性が向上すること
は明らかである。Example 9 Measurement of Ice Crystallization Inhibitory Activity The ice crystallization inhibitory activity of each of the extracts obtained in Example 8 and Comparative Example 3 was measured in the same manner as described in Example 3. As a result of quantifying the average area of the ice crystals in the obtained image as a ratio to the average area of the ice crystals obtained for the aqueous solution of the extract of Comparative Example 3, the value was 0.88, which was extracted after drying. The average area of ice crystals in the aqueous solution of the extract thus obtained (Example 8) was smaller than the average area of ice crystals in the aqueous solution of the extract of Comparative Example 3 that was not subjected to drying treatment. Also from this result, it is clear that the ice crystallization inhibitory activity of the ice crystallization substance in the mustard sprout is improved by drying it after induction of the ice crystallization inhibitory activity by low temperature acclimation.
実施例10 低温馴化と乾燥後に抽出し、さらに吸着処理した抽出物の製造
実施例8で得られたタンパク質濃度10mg/mLの抽出液(22mL)に活性炭(二村化学工業社製,太閤FC,220mg)を添加し、150rpmにて30分間振とうしてこれを混合した。遠心分離機(日立ハイテクノロジーズ社製、品番:CR22G)により10,000×gで30分間遠心分離して活性炭を除去し、上清(19mL)を得た。Example 10 Manufacture of an extract that was extracted after low temperature acclimation and drying, and further subjected to adsorption treatment The activated carbon (Nimura FC, 220 mg) was added to the extract (22 mL) with a protein concentration of 10 mg / mL obtained in Example 8. ) Was added and mixed by shaking at 150 rpm for 30 minutes. The activated carbon was removed by centrifugation at 10,000 × g for 30 minutes using a centrifuge (manufactured by Hitachi High-Technologies Corporation, product number: CR22G) to obtain a supernatant (19 mL).
実施例11 タンパク質濃度および氷結晶化阻害活性の測定
実施例10で得た抽出液について、タンパク質濃度をBCA法で測定した。また、実施例3に記載の方法と同様に氷結晶化阻害活性を測定した。この結果を実施例8の活性炭処理前の抽出液と比較して表3に示す。表3の氷結晶化阻害活性の数値は、それぞれの溶液を測定して得られた画像中の氷結晶の平均面積を、水を試料として同様に測定したときの氷結晶の平均面積に対する比として数値化した相対値である。この数値が小さいほど氷結晶化阻害活性が強いことを示している。Example 11 Measurement of Protein Concentration and Ice Crystallization Inhibitory Activity For the extract obtained in Example 10, the protein concentration was measured by the BCA method. Further, the ice crystallization inhibitory activity was measured in the same manner as in the method described in Example 3. The results are shown in Table 3 in comparison with the extract of Example 8 before the activated carbon treatment. The numerical value of ice crystallization inhibitory activity in Table 3 is the ratio of the average area of ice crystals in the image obtained by measuring each solution to the average area of ice crystals when water was used as a sample. It is a relative value that is digitized. It shows that ice crystallization inhibitory activity is so strong that this figure is small.
表3に示すように、マスタードスプラウトを乾燥した後に抽出して得られた抽出液を、さらに活性炭で処理すると、溶液中のタンパク質の約86%が除去された。一方で、氷結晶の平均面積は殆ど変化しておらず、溶液の氷結晶化阻害活性は維持されていた。この結果から、低温馴化による氷結晶化阻害活性誘導後のマスタードスプラウトを、乾燥処理した後に抽出し、さらにこれを活性炭で処理することによって、氷結晶化阻害物質の精製度が著しく向上した抽出物を得ることができることは明らかである。 As shown in Table 3, when the extract obtained after drying the mustard sprout was further treated with activated carbon, about 86% of the protein in the solution was removed. On the other hand, the average area of ice crystals was hardly changed, and the ice crystallization inhibitory activity of the solution was maintained. From this result, extract of mustard sprout after induction of ice crystallization inhibitory activity by low-temperature acclimation was extracted after drying, and further treated with activated charcoal to significantly improve the purity of ice crystallization inhibitor. It is clear that can be obtained.
実施例12 低温馴化と乾燥後の抽出による抽出物の製造
市販のレッドキャベツスプラウトを4℃にて低温馴化し、氷結晶化阻害物質を誘導した。馴化4日目のスプラウト(20g)を金属製トレーに取り、実施例2に記載の方法と同様にして40℃で温風乾燥し、乾燥物(0.8g)を得た。この乾燥物に水(80mL)を加え、50℃で2時間加熱抽出した後に濾液を乾燥し、抽出物(389mg)を得た。この抽出物を、その濃度が100mg/mLとなるよう水に溶解し、抽出液を得た。Example 12 Preparation of extract by low-temperature acclimation and extraction after drying Commercial red cabbage sprout was acclimated at 4 ° C. to induce an ice crystallization inhibitor. The sprout (20 g) of acclimatization day 4 was taken in a metal tray and dried in warm air at 40 ° C. in the same manner as described in Example 2 to obtain a dried product (0.8 g). Water (80 mL) was added to the dried product and the mixture was heated and extracted at 50 ° C. for 2 hours, and then the filtrate was dried to obtain an extract (389 mg). This extract was dissolved in water so as to have a concentration of 100 mg / mL to obtain an extract.
比較例4
実施例12で低温馴化したレッドキャベツスプラウト(20g)を乾燥せず、そのまま水(80mL)を加え、50℃で2時間加熱抽出し、抽出物(390mg)を得た。この抽出物を、その質濃度が100mg/mLとなるように水に溶解し、抽出液を得た。Comparative Example 4
The red cabbage sprout (20 g) acclimated to low temperature in Example 12 was not dried, water (80 mL) was added as it was, and the mixture was extracted by heating at 50 ° C. for 2 hours to obtain an extract (390 mg). This extract was dissolved in water so that the quality concentration was 100 mg / mL to obtain an extract.
実施例13 低温馴化と乾燥後の抽出による抽出物の製造
市販のケールスプラウトを4℃にて低温馴化し、氷結晶化阻害物質を誘導した。馴化4日目のスプラウト(20g)を金属製トレーに取り、実施例2に記載の方法と同様にして40℃で温風乾燥し、乾燥物(1.2g)を得た。この乾燥物に水(80mL)を加え、50℃で2時間加熱抽出した後に濾液を乾燥し、抽出物(577mg)を得た。この抽出物を、その濃度が100mg/mLとなるように水に溶解し、抽出液を得た。Example 13 Preparation of extract by low temperature acclimation and extraction after drying A commercial kale sprout was cold conditioned at 4 ° C. to induce an ice crystallization inhibitor. The sprout (20 g) of acclimatization day 4 was placed in a metal tray and dried in warm air at 40 ° C. in the same manner as described in Example 2 to obtain a dried product (1.2 g). Water (80 mL) was added to the dried product, followed by extraction with heating at 50 ° C. for 2 hours, and then the filtrate was dried to obtain an extract (577 mg). This extract was dissolved in water so as to have a concentration of 100 mg / mL to obtain an extract.
比較例5
実施例13で低温馴化したケールスプラウト(20g)を乾燥せず、そのまま水(80mL)を加え、50℃で2時間加熱抽出し、抽出物(559mg)を得た。この抽出物を、その質濃度が100mg/mLとなるように水に溶解し、抽出液を得た。Comparative Example 5
The kale sprout (20 g) conditioned at low temperature in Example 13 was not dried, water (80 mL) was added as it was, and the mixture was extracted by heating at 50 ° C. for 2 hours to obtain an extract (559 mg). This extract was dissolved in water so that the quality concentration was 100 mg / mL to obtain an extract.
実施例14 氷結晶化阻害活性の測定
実施例12、13および比較例4、5で得た抽出物の水溶液について、それぞれ実施例3に記載の方法と同様に氷結晶化阻害活性を測定した。実施例12、13の抽出液で得られた画像中の氷結晶の平均面積を、それぞれ比較例4、5の抽出物の抽出液について得られた氷結晶の平均面積に対する比として数値化した。この結果を表4に示す。Example 14 Measurement of Ice Crystallization Inhibitory Activity With respect to the aqueous solutions of the extracts obtained in Examples 12 and 13 and Comparative Examples 4 and 5, ice crystallization inhibitory activity was measured in the same manner as described in Example 3. The average area of ice crystals in the images obtained with the extracts of Examples 12 and 13 was quantified as a ratio to the average area of ice crystals obtained for the extracts of the extracts of Comparative Examples 4 and 5, respectively. The results are shown in Table 4.
表4に示すように、乾燥後に抽出して得られる抽出物の水溶液(実施例12、13)の氷結晶の平均面積は、乾燥処理を施していない比較例4、5の抽出物の水溶液の氷結晶の平均面積よりも小さくなっていた。この結果からも、低温馴化による氷結晶化阻害活性の誘導後に乾燥することによって、スプラウト中の氷結晶化阻害物質の活性が向上することは明らかである。 As shown in Table 4, the average area of the ice crystals of the aqueous solution of the extract obtained by extraction after drying (Examples 12 and 13) is that of the aqueous solution of the extract of Comparative Examples 4 and 5 that has not been subjected to the drying treatment. It was smaller than the average area of ice crystals. Also from this result, it is clear that the activity of the ice crystallization inhibitor in the sprout is improved by drying after induction of the ice crystallization inhibitory activity by low temperature acclimation.
実施例15 低温馴化後に抽出し、さらに吸着処理した抽出物の製造
(1) 低温馴化後の抽出
市販のマスタードスプラウト(村上農園製)を15℃にて低温馴化し、氷結晶化阻害活性を誘導した。馴化10日目のマスタードスプラウト(50g)に水(100mL)を加え、105℃、0.12MPaで20分間加圧抽出した。濾紙(アドバンテック社製,保留粒子径:5μm)を用いて濾過した後、回収した濾液を減圧濃縮装置(東京理化機器製ロータリーエバポレーター,品番:N−1000−S−W)で減圧濃縮した。さらに、得られた濃縮液を遠心分離機(日立ハイテクノロジーズ社製,品番:CR22G)により18,000×gで20分間遠心分離し、上清(80mL)を得た。この抽出液のタンパク質濃度を、Bio−Rad社製のProtein Assay キットを用いたBradford法で測定したところ、1.04mg/mLであった。Example 15 Production of extract extracted after low temperature acclimation and further subjected to adsorption treatment (1) Extraction after low temperature acclimation Commercially mustard sprout (manufactured by Murakami Farm) was acclimated at 15 ° C. to induce ice crystallization inhibitory activity. did. Water (100 mL) was added to mustard sprout (50 g) on day 10 of acclimation, and pressure extraction was performed at 105 ° C. and 0.12 MPa for 20 minutes. After filtration using a filter paper (manufactured by Advantech Co., Ltd., retained particle size: 5 μm), the collected filtrate was concentrated under reduced pressure using a vacuum concentrator (rotary evaporator manufactured by Tokyo Rika Co., Ltd., product number: N-1000-SW). Furthermore, the obtained concentrated liquid was centrifuged at 18,000 × g for 20 minutes with a centrifuge (manufactured by Hitachi High-Technologies Corporation, product number: CR22G) to obtain a supernatant (80 mL). The protein concentration of this extract was measured by the Bradford method using a Protein Assay kit manufactured by Bio-Rad, and found to be 1.04 mg / mL.
(2) 吸着処理
上記抽出液(1.0mL)を1.5mLチューブに取り、粉末状活性炭(二村化学工業株式会社製,太閤FC)を、12.5mg、25.0mgまたは50.0mg加えて攪拌・混合した。また、対照として、上記抽出液に活性炭を加えずに同様に攪拌・混合した。それぞれ遠心分離機(日立ハイテクノロジーズ社製,品番:CR22G)により10,000×gで30分間遠心分離して活性炭を除去した上清を回収した。得られた上清をメンブレンフィルター(アドバンテック社製,cellurose acetate,径:0.45μm)に通して得られた活性炭処理溶液を、以下のタンパク濃度測定及び氷結晶化阻害活性測定に供した。(2) Adsorption treatment Take the above extract (1.0 mL) in a 1.5 mL tube, add 12.5 mg, 25.0 mg or 50.0 mg of powdered activated carbon (Nimura Chemical Co., Ltd., Dazai FC). Stir and mix. As a control, the above extract was similarly stirred and mixed without adding activated carbon. Each supernatant was removed by centrifuging at 10,000 × g for 30 minutes with a centrifuge (manufactured by Hitachi High-Technologies Corporation, product number: CR22G) to remove the activated carbon. The activated carbon-treated solution obtained by passing the obtained supernatant through a membrane filter (manufactured by Advantech, cellulose acetate, diameter: 0.45 μm) was subjected to the following protein concentration measurement and ice crystallization inhibition activity measurement.
実施例16 タンパク濃度及び氷結晶化阻害活性の測定
実施例15で得たそれぞれの活性炭処理溶液および対照の溶液について、タンパク質濃度をBradford法で測定した。Example 16 Measurement of Protein Concentration and Ice Crystallization Inhibitory Activity The protein concentration of each activated carbon treatment solution obtained in Example 15 and the control solution was measured by the Bradford method.
また、氷結晶化阻害活性の測定を以下の方法で行った。実施例15で得たそれぞれの溶液を、60w/v%のショ糖溶液と1:1(v/v)の割合で混合した。得られた混合液(1μL)をカバーガラスで挟み込んだ。加熱冷却載物台(LINKAM社製,LK‐600PM)を備えた光学顕微鏡(OLYMPUS社製,BX50)のガラスシャーレを20℃に保ち、その上に上記カバーガラスを載せて、100℃/minの速度で−40℃まで冷却した。次に、100℃/minの速度で−6℃まで温度を上げた。−6℃になった時を0分として、その後、光学顕微鏡をそのままの状態に保ち、30分後の画像を取り込んだ。得られた画像中に存在する氷結晶の平均面積を算出し、これを氷結晶化阻害活性の指標とした。この結果を表5に示す。表5中の氷結晶化阻害活性の値は、それぞれの溶液を測定して得られた画像中の氷結晶の平均面積を、30w/v%のショ糖溶液を用いて同様に測定したときの氷結晶の平均面積に対する比として数値化した相対値を算出し、さらにその逆数を活性値として、これをタンパク質濃度で除した数値を、対照の活性値に対する比として数値化したものである。この数値が大きいほど氷結晶化阻害物質の比活性が強いことを示している The ice crystallization inhibitory activity was measured by the following method. Each solution obtained in Example 15 was mixed with a 60 w / v% sucrose solution at a ratio of 1: 1 (v / v). The obtained mixed liquid (1 μL) was sandwiched between cover glasses. Keep the glass petri dish of an optical microscope (OLYMPUS, BX50) equipped with a heating / cooling stage (LINKAM, LK-600PM) at 20 ° C., and place the cover glass on top of the glass dish. Cooled to -40 ° C at a rate. Next, the temperature was increased to −6 ° C. at a rate of 100 ° C./min. The time when the temperature reached −6 ° C. was set to 0 minute, and then the optical microscope was kept as it was, and the image after 30 minutes was captured. The average area of ice crystals present in the obtained image was calculated and used as an index of ice crystallization inhibitory activity. The results are shown in Table 5. The value of the ice crystallization inhibitory activity in Table 5 is obtained when the average area of ice crystals in the image obtained by measuring each solution was measured in the same manner using a 30 w / v% sucrose solution. The relative value calculated as a ratio to the average area of ice crystals is calculated, and the reciprocal number is calculated as the activity value, and the value obtained by dividing this by the protein concentration is expressed as the ratio to the control activity value. The larger this value, the stronger the specific activity of the ice crystallization inhibitor.
表1に示すように、マスタードスプラウト抽出液のタンパク質濃度は活性炭添加量に応じて著しく減少するが、氷結晶化阻害物質の比活性は著しく増大した。この結果から、マスタードスプラウト抽出液を活性炭で処理することにより、その氷結晶化阻害活性を損なうことなく溶液中の不要分を除去し、氷結晶化阻害物質の精製度を著しく向上できることは明らかである。 As shown in Table 1, the protein concentration of the mustard sprout extract decreased markedly depending on the amount of activated carbon added, but the specific activity of the ice crystallization inhibitor increased significantly. From this result, it is clear that treating the mustard sprout extract with activated carbon can remove unnecessary components in the solution without impairing its ice crystallization inhibitory activity and significantly improve the purity of the ice crystallization inhibitor. is there.
実施例17 低温馴化後に抽出し、さらに吸着処理した抽出物の製造
(1) 低温馴化後の抽出
市販のマスタードスプラウトを15℃にて低温馴化し、氷結晶化阻害物質を誘導した。馴化10日目のマスタードスプラウト(50g)に水(100mL)を加え、実施例1に記載の方法と同様にして、105℃、0.12MPaで20分間加圧抽出した。得られた抽出液を減圧濃縮装置で濃縮し、水を除去してこれを固形化することにより抽出物(880mg)を得た。この固形の抽出物を、そのタンパク質濃度がBCA法により10mg/mLとなるように水に再度溶解し、抽出液(26mL)を得た。Example 17 Production of Extract Extracted after Low Temperature Conditioning and Adsorption Treatment (1) Extraction after Low Temperature Conditioning Commercially mustard sprout was acclimatized at 15 ° C. to induce an ice crystallization inhibitor. Water (100 mL) was added to the mustard sprout (50 g) on day 10 of acclimation, and pressure extraction was performed at 105 ° C. and 0.12 MPa for 20 minutes in the same manner as described in Example 1. The obtained extract was concentrated with a vacuum concentrator, water was removed, and this was solidified to obtain an extract (880 mg). This solid extract was dissolved again in water so that the protein concentration became 10 mg / mL by the BCA method to obtain an extract (26 mL).
(2) 吸着処理
上記抽出液(1.0mL)を1.5mLチューブに取り、これに活性炭(二村化学工業株式会社製,太閤FC)を、2.5mg、5.0mgまたは10.0mg加え、攪拌・混合した。また、対照として、上記抽出液に活性炭を加えずに同様に攪拌・混合した。それぞれ10,000×gで30分間遠心分離して活性炭を除去した上清を回収した。(2) Adsorption treatment Take the above extract (1.0 mL) in a 1.5 mL tube, add 2.5 mg, 5.0 mg, or 10.0 mg of activated carbon (Futamura Chemical Co., Ltd., Dazai FC) to this, Stir and mix. As a control, the above extract was similarly stirred and mixed without adding activated carbon. Centrifugation was performed at 10,000 × g for 30 minutes to recover the supernatant from which the activated carbon had been removed.
実施例18 タンパク質濃度及び氷結晶化阻害活性の測定
実施例17で得たそれぞれの上清について、タンパク質濃度をBCA法で測定した。また、実施例16に記載の方法と同様に氷結晶化阻害活性を測定した。この結果を表6に示す。表6のタンパク質濃度の数値は、対照の溶液について得られたタンパク質濃度に対する比として数値化した。また表6の氷結晶化阻害活性の数値は、それぞれの溶液を測定して得られた画像中の氷結晶の平均面積を、30w/v%のショ糖溶液を用いて同様に測定したときの氷結晶の平均面積に対する比として数値化した相対値を算出し、さらにその逆数を活性値として、これをタンパク質濃度で除した数値を、対照の活性値に対する比として数値化したものである。この数値が大きいほど氷結晶化阻害物質の比活性が強いことを示している。Example 18 Measurement of Protein Concentration and Ice Crystallization Inhibitory Activity For each supernatant obtained in Example 17, the protein concentration was measured by the BCA method. Further, the ice crystallization inhibitory activity was measured in the same manner as in the method described in Example 16. The results are shown in Table 6. The protein concentration values in Table 6 were quantified as a ratio to the protein concentration obtained for the control solution. Moreover, the numerical values of the ice crystallization inhibitory activity in Table 6 are the values obtained when the average area of ice crystals in the image obtained by measuring each solution was measured in the same manner using a 30 w / v% sucrose solution. The relative value calculated as a ratio to the average area of ice crystals is calculated, and the reciprocal number is calculated as the activity value, and the value obtained by dividing this by the protein concentration is expressed as the ratio to the control activity value. The larger this value, the stronger the specific activity of the ice crystallization inhibitor.
表6に示すように、マスタードスプラウト抽出液のタンパク質濃度は活性炭添加量に応じて大きく減少するが、氷結晶化阻害物質の比活性は増大した。この結果から、マスタードスプラウト抽出液を活性炭で処理することにより、その氷結晶化阻害物質の精製度を向上できることは明らかである。 As shown in Table 6, the protein concentration of the mustard sprout extract decreased greatly with the amount of activated carbon added, but the specific activity of the ice crystallization inhibitor increased. From this result, it is clear that the degree of purification of the ice crystallization inhibitor can be improved by treating the mustard sprout extract with activated carbon.
実施例19 低温馴化後に抽出し、さらに吸着処理した抽出物の製造
上記実施例17(1)で得た抽出液(1.0mL)を1.5mLチューブに取り、これにシリカゲル(和光純薬工業社製,ワコーゲルC−200,200mg)を加えて攪拌・混合した。遠心分離によりシリカゲルを除去した上清を回収した。Example 19 Manufacture of extract extracted after acclimation at low temperature and further subjected to adsorption treatment The extract (1.0 mL) obtained in Example 17 (1) above was placed in a 1.5 mL tube, and silica gel (Wako Pure Chemical Industries, Ltd.) (Wakogel C-200, 200 mg) was added and stirred and mixed. The supernatant from which the silica gel was removed by centrifugation was collected.
実施例20 低温馴化後に抽出し、さらに吸着処理した抽出物の製造
上記実施例17(1)で得た抽出液(1.0mL)を1.5mLチューブに取り、これにアクリル系合成吸着体であるダイヤイオンHP2MG(三菱化学株式会社製,200mg)を加えて攪拌・混合した。遠心分離により吸着体を除去した上清を回収した。Example 20 Manufacture of extract extracted after acclimation at low temperature and further subjected to adsorption treatment The extract (1.0 mL) obtained in Example 17 (1) above was placed in a 1.5 mL tube, and this was mixed with an acrylic synthetic adsorbent. A certain Diaion HP2MG (Mitsubishi Chemical Corporation, 200 mg) was added and stirred and mixed. The supernatant from which the adsorbent had been removed by centrifugation was collected.
実施例21 低温馴化後に抽出し、さらに吸着処理した抽出物の製造
上記実施例17(1)で得た抽出液(1.0mL)を1.5mLチューブに取り、これに陽イオン交換樹脂であるダイヤイオンWK20(三菱化学株式会社製,200mg)を加えて攪拌・混合した。遠心分離により吸着体を除去した上清を回収した。Example 21 Manufacture of extract extracted after acclimation at low temperature and further subjected to adsorption treatment The extract (1.0 mL) obtained in Example 17 (1) above was taken in a 1.5 mL tube, and this was a cation exchange resin. Diaion WK20 (Mitsubishi Chemical Corporation, 200 mg) was added and stirred and mixed. The supernatant from which the adsorbent had been removed by centrifugation was collected.
比較例6
上記実施例17(1)で得た抽出液(1.0mL)を1.5mLチューブに取り、実施例19〜21と同様にしてこれを遠心分離し、上清を回収した。Comparative Example 6
The extract (1.0 mL) obtained in Example 17 (1) was taken in a 1.5 mL tube, and centrifuged in the same manner as in Examples 19 to 21, and the supernatant was collected.
実施例22 タンパク質濃度及び氷結晶化阻害活性の測定
実施例19〜21および比較例6で得たそれぞれの上清について、タンパク質濃度をBCA法で測定した。また、実施例16に記載の方法と同様に氷結晶化阻害活性を測定した。この結果を表7に示す。表7のタンパク質濃度の数値は、比較例6の溶液について得られたタンパク質濃度に対する比として数値化した。また表7の氷結晶化阻害活性の数値は、それぞれの溶液を測定して得られた画像中の氷結晶の平均面積を、30w/v%のショ糖溶液を用いて同様に測定したときの氷結晶の平均面積に対する比として数値化した相対値を算出し、さらにその逆数を活性値として、これをタンパク質濃度で除した数値を、比較例6の活性値に対する比として数値化したものである。この数値が大きいほど氷結晶化阻害物質の比活性が強いことを示している。Example 22 Measurement of protein concentration and ice crystallization inhibitory activity For each supernatant obtained in Examples 19 to 21 and Comparative Example 6, the protein concentration was measured by the BCA method. Further, the ice crystallization inhibitory activity was measured in the same manner as in the method described in Example 16. The results are shown in Table 7. The protein concentration values in Table 7 were quantified as a ratio to the protein concentration obtained for the solution of Comparative Example 6. In addition, the numerical values of the ice crystallization inhibitory activity in Table 7 are the values obtained when the average area of ice crystals in the image obtained by measuring each solution was measured in the same manner using a 30 w / v% sucrose solution. The relative value calculated as a ratio to the average area of ice crystals was calculated, and the reciprocal of the calculated value was divided by the protein concentration, and the numerical value obtained by dividing the value by the protein concentration was expressed as the ratio to the activity value of Comparative Example 6. . The larger this value, the stronger the specific activity of the ice crystallization inhibitor.
表7に示すように、マスタードスプラウト抽出液のタンパク質濃度は実施例19〜21の各吸着体による処理で大きく減少するが、氷結晶化阻害物質の比活性はいずれの吸着体処理においても増大した。この結果から、マスタードスプラウト抽出液をこれらの吸着体で処理することにより、その氷結晶化阻害物質の精製度を向上できることは明らかである。 As shown in Table 7, the protein concentration of the mustard sprout extract was greatly reduced by the treatment with each of the adsorbents of Examples 19 to 21, but the specific activity of the ice crystallization inhibitor increased in any of the adsorbent treatments. . From this result, it is clear that the degree of purification of the ice crystallization inhibitor can be improved by treating the mustard sprout extract with these adsorbents.
本発明に係る氷結晶化阻害物質を含む植物抽出物の第一の製造方法によれば、より一層優れた氷結晶化阻害活性を示す植物抽出物を効率良く製造できるので、優れた氷結晶化阻害活性を有する組成物を提供できるという効果を奏する。また、本発明に係る氷結晶化阻害物質を含む植物抽出物の第二の製造方法によれば、氷結晶化阻害物質の精製度を著しく向上させることができるので、優れた氷結晶化阻害活性を有する組成物を提供できるという効果を奏する。よって、本発明の製造方法を用いることにより、実用にかなう優れた氷結晶化阻害活性を有する氷結晶化阻害物質を、食品製造に利用できる安全な工程で、簡単に、効率よく、安価に提供することが可能になる。 According to the first method for producing a plant extract containing an ice crystallization inhibitor according to the present invention, it is possible to efficiently produce a plant extract exhibiting further excellent ice crystallization inhibitory activity. There exists an effect that the composition which has inhibitory activity can be provided. Further, according to the second method for producing a plant extract containing an ice crystallization inhibitor according to the present invention, the purification degree of the ice crystallization inhibitor can be remarkably improved, so that the ice crystallization inhibitory activity is excellent. There exists an effect that the composition which has can be provided. Therefore, by using the production method of the present invention, an ice crystallization inhibitor having excellent ice crystallization inhibitory activity suitable for practical use can be provided simply, efficiently and inexpensively in a safe process that can be used for food production. It becomes possible to do.
Claims (25)
氷結晶化阻害物質を含む植物を乾燥する工程;および
乾燥した植物から氷結晶化阻害物質を抽出する工程;
を含むことを特徴とする製造方法。A method for producing a plant extract comprising an ice crystallization inhibitor comprising:
Drying the plant containing the ice crystallization inhibitor; and extracting the ice crystallization inhibitor from the dried plant;
The manufacturing method characterized by including.
植物から氷結晶化阻害物質を抽出する工程;および
抽出物を吸着体で処理する工程;
を含むことを特徴とする製造方法。A method for producing a plant extract containing an ice crystallization inhibitor, the method comprising extracting the ice crystallization inhibitor from a plant; and treating the extract with an adsorbent;
The manufacturing method characterized by including.
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